Bone-like prosthetic implants

ABSTRACT

A prosthetic implant comprising a biocompatible three-dimensional scaffold and at least two cell types selected from the group consisting of osteoblasts, osteoclasts, and endothelial cells or progenitors thereof.

FIELD OF THE INVENTION

The present invention relates to the field of bone, cartilage and hardtissue prosthetics, and more particularly to the use of cellular-basedimplants for the preparation of prosthetic implants for bone replacementand repair, cartilage replacement and repair, and for other hard tissueapplications.

BACKGROUND OF THE INVENTION

The repair of outsized deficiencies, typically defined as gaps of atleast about 2.4 mm in size, in the diaphyseal, craniomaxillofacial andother skeletal bones is a considerable problem in orthopedic surgery.

In 1998, about 300,000 bone-graft procedures were performed in theUnited States alone. This number increased to approximately 450,000 bythe year of 2000, when the number of bone grafting procedures performedworldwide exceeded 2.2 million (Lewandrowski et al, 2000). Of the300,000 procedures performed in 1998, 90% involved the use of eitherautologous grafts (i.e. using tissue from another part of the body ofthe patient), or of allografts (i.e. using tissue from a live humandonor or cadaver). Therefore, a phase of tissue harvest from the patientor from a donor is required.

The tissue harvesting is executed by a surgical procedure usuallyinvolved collecting tissue from the iliac crest, the distal femur, theproximal tibia, the fibula, or from other small bones. The harvestedtissue is restructured and transplanted at the damaged site.

However, the graft-harvesting procedures are associated withconsiderable morbidity and substantial pain. Tissue harvesting for anautologous grafts or from live donors for an allograft may also resultin complications such as inflammation, infection, or even death.Allografts taken from live donors or cadavers also carry risks ofdisease transmission and although grafts are subjected to protective andsterilization treatments such as tissue freezing, freeze-drying, gammairradiation, electron beam radiation, and ethylene oxide, this risk isnot completely removed. Furthermore, substantial supply problems exist,as the bone tissues harvested are limited.

The limited supply and inherited harvesting complications have inspiredthe development of alternative strategies for the repair of significantbone defects.

The use of 3-dimensional (3-D) bone substitutes such as bone extract,polymer or mineral scaffolds as implants has been investigated andporous biocompatible scaffolds have been used for the repair andregeneration of bone tissue.

Early attempts at tissue repair have focused mainly on the use ofamorphous, biocompatible foam as porous plugs to fill large voids inbone. U.S. Pat. No. 4,186,448 described the use of porous mesh plugscomposed of polyhydroxy acid polymers, such as polylactide, for healingbone voids. Several different methods for making other scaffolds werealso described (i.e. U.S. Pat. Nos. 5,133,755; 5,514,378; 5,522,895;5,607,474; 5,677,355; 5,686,091; 5,716,413; 5,755,792; 5,769,899;5,770,193; 6,333,029; 6,365,149 and 6,534,084).

Bone marrow (BM) has been shown to contain population of cells thatpossess osteogenic potential. As such, an alternative to thescaffold-osteoinductive approach is to transplant into patients livingcells that possess this capacity.

Cytokine-manipulated, naïve autologous and allogeneic BM cells havesuccessfully healed diffracted or resorbed bones in experimental models(Werntz et al, 1996; Lane et al, 1999; Nilsson et al, 1999; Kawaguchi etal, 2004) and human patients (Horwitz et al, 1999; Horwitz et al 2001,2004).

These techniques were further developed by using enriched mesenchymalcells for transplantation, and were demonstrated to be successful inanimal models and human patients (Pereira et al, 1995; Shang et al,2001; Horwitz et al, 2002). Accordingly, U.S. Pat. Nos. 5,716,616 and6,200,606 describe experimental therapies for treating bone losssyndromes that are based on the implantation of fresh stromal cellsisolated from autologous or syngeneic individuals to recipients (i.e.U.S. Pat. No. 5,716,616). Although this approach is promising in theory,it is difficult in practice to obtain the sufficient quantities of BMhaving the requisite number of osteoprogenitor cells.

Tissue Induction methods (TI) have been developed, wherein tissueregeneration occurs through in-growth of surrounding cells into 3-Dscaffolds. The limitations of the TI procedure include the requirementfor scaffolding material that possesses both TI capability andmechanical properties similar to those of autologous bone tissues.

Another approach to bone tissue generation is referred to as “complexcell transplantation”, which combines scaffold technology with cellcultivation techniques. In its simplest form, autologous BM aspirate ispassed through a biocompatible, implantable substrate placedintra-peritoneally to provide a composite bone graft (Nade et al, 1983and U.S. Pat. Nos. 5,824,084; 6,049,026).

Alternatively, progenitor cells of the osteogenic lineage are seededonto biocompatible (biodegradable or non-biodegradable) scaffolds in thepresence or absence of growth promoting factors (U.S. Pat. Nos.6,541,024; 6,544,290; 6,852,330). Transplantation into affected patientsis performed following an ex-vivo expansion phase of the cells on thegiven scaffold. Using this approach, either primary osteogenic cells orexpanded Mesenchymal Stromal Cells (MSC) layered upon ceramic scaffoldswas able to regenerate bone tissue (Kadiyala et al, 1997; Bruder et al,1998a,b; Cinotti et al, 2004).

However, experimental results revealed a number of disadvantages ofthose complex cell transplantations. Firstly, bone marrowtransplantation (BMT) in human patients is associated with a generaldecrease in the skeletal mineral density (Valimaki et al, 1999).Secondly, it was demonstrated that after BMT, although peripheralmononuclear cells (MNC) in the recipients are of donor origin, the BMstroma cells and MSCs are basically of the recipient origin (Koc et al,1999; Lee et al, 2002). Finally, during the first year follow-up of bonemarrow transplanted patients, a gradual decrease in bone repair wasevident and significant loss of donor MSC was observed (Lee et al,2002).

Living bone is a continuously evolving organ and in the normal course ofbone maintenance, a constant remodeling process is being employed. Inthose procedures, Old bone is being replaced by new bone and the organresponds to its environment changing requirements for strength andelasticity. Therefore, normal remodeling progression requires that themechanical loading processes of bone resorption and bone formationprocedures are tightly coordinated.

In cellular terms, this depends on sequential functioning of osteoclasts(bone resorbing cells) and osteoblasts (bone forming cells). Inaddition, endothelial cell and endothelial cell precursors (angioblasts)are required to form the new blood vessels in the developed bone tissue.Yet, the various cell types participating in bone formation are ofdifferent lineages. It is now known that osteoblasts stalk frommesenchymal stem cells, while osteoclasts (directly originating fromHematopoietic Stem Cells (HSC)) and endothelial cells are descendents ofa common blast colony-forming cell (Choi et al, 1998; Hamaguchi et al,1999). As such, methodologies for ex-vivo production of bone-likematerial that rely on osteoblasts as the exclusive cellular componentsuffer from an inherited fault.

It would be highly advantageous to have a material for use in repairingbone lesions that is devoid of at least some of the limitations of theprior art.

External fixation devices for keeping fractured bones stabilized andaligned, and ensuring that the bones remain at an optimal positionduring the healing process are known and commonly used. Such devicestypically comprise a plurality of pins placed proximal and distal to thefracture, fixed in a surrounding external mechanical assembly.

External fixation devices are also used for reconstructive orthopedics,such as treatment of bone losses and defects. In such cases the devicecan either remain in place until healing occurs and then to be removed,leaving no foreign material inside the bone, or it can totally orpartially remain inside the bone.

External fixation devices are further useful in experimental models ofbone repair which have been developed for a variety of purposes,including the investigation of factors influencing fracture repair, anddevelopment of improved methods of managing fractures in human andanimals. Such models make use of long bones of large experimentalanimals, whose weight is more than 40 gr, such as dogs, sheep, rabbitsand rats. The term long bone refers to bones in which the length isgreater than the width, such as a femur, a tibia, a humerus and aradius.

US application No. 20030149437 to the same inventors as the presentapplication discloses a method of repairing a long bone having a defect.The method comprising mechanically fixating the long bone or portionsthereof and filling the defect with a biodegradable scaffold impregnatedwith growth factors and/or cells to cause a regeneration of the bone.

SUMMARY OF THE INVENTION

The present invention provides an ideal solution for the ex-vivoregeneration of remodeled bone, cartilage and other hard tissueapplications. The background art describes bone substitutes made fromcells of osteogenic lineage cultured on 3-D scaffold. In contrast withthis art, the present invention manipulates co-culture and multi-cellcultures made up of two or more independent cell types growing on 3-Dscaffolds to optimize the bone regeneration and remodeling processes,preferably through a flow system and more preferably to grow cells at ahigh density.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepatent specification, including definitions, will control.

As used herein the phrase “three dimensional cultures” refers tocultures in which the cells are disposed to conditions which arecompatible with cell growth while allowing the cells to grow in morethan one layer. It is well appreciated that the in situ environment ofthe cells in living organism (or a tissue) is in a three dimensionalarchitecture. Cells are surrounded by other cells. They are held in acomplex network of extra cellular matrix nanoscale fibers that allowsthe establishment of various local microenvironments. Their extracellular ligands mediate not only the attachment to the basal membranebut also access to a variety of vascular and lymphatic vessels. Oxygen,hormones and nutrients are ferried to cells and waste products arecarried away. The conditions in the three dimensional culture of theinvention are designed to mimic such an environment as is furtherexemplified below. Thus, the three-dimensional scaffold of the presentinvention is differed and preferred to any type of two-dimensionalartificial cell environments.

It will be appreciated that the conditions of the three-dimensionalculture are such that enable the expansion of the adherent cells.

As used herein the terms “expanding” and “expansion” refer tosubstantially differentiation-less maintenance of the cells andultimately cell growth, i.e., increase of a cell population (e.g., atleast 2 fold) without differentiation accompanying such increase.

As used herein the terms “maintaining” and “maintenance” refer tosubstantially differentiation-less cell renewal, i.e., substantiallystationary cell population without differentiation accompanying such astationary state.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, integers, steps or components but do not preclude the additionof one or more additional features, integers, steps, components orgroups thereof. These terms encompass the terms “consisting of” and“consisting essentially of”.

The phrase “consisting essentially of or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

According to some embodiments, there is provided a prosthetic implantcomprising a biocompatible three-dimensional scaffold and at least twocell types selected from the group consisting of osteoblasts,osteoclasts, chondrocytes and endothelial cells or progenitors thereof.Optionally, the osteoblasts are derived from mesenchymal stem cells.Also optionally, the at least two cell types are obtained from a sourceselected from the group consisting of an autologous source, a syngeneicsource and an allogeneic source. Preferably, the source comprises one ormore of bone marrow, placenta, adipose tissue, cord blood, peripheralblood, mobilized peripheral blood, embryonic stem cells and the like.

Optionally the implant further comprises at least three different celltypes selected from the group consisting of osteoblasts, osteoclasts,and endothelial cells or progenitors thereof. The progenitors arepreferably selected from the group consisting of mesenchymal stromalcells, hematopoietic stem cells, and angioblasts.

Also optionally, the at least two cell types are selected from the groupconsisting of hematopoietic cells, vascular endothelial cells andmesenchymal cells and their progenitors.

Preferably, the 3-dimensional scaffold comprises a preshaped andsponge-like or porous and can e.g. comprise an organic, ceramic ormetallic material, or comprises dry, frozen or dematerialized boneparticles or comprises a polymer selected from the group consisting ofaliphatic polyesters, poly(amino acids), co-poly(ether-esters),polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates,poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters,polyoxaesters containing amine groups, poly(anhydrides),polyphosphazenes, bioactive glass, calcium phosphate derivatives,calcium sulfate derivatives, calcium hydroxyapeptite, silicate matrices,hydroxyapeptite, hyalauronic acid, beta-3 calcium phosphate,cross-linked collage fibers, resin based materials and combinationsthereof.

More preferably, the 3-dimensional scaffold comprises a solid or gellike biodegradable polymer.

Optionally and most preferably, the biodegradable polymer is a naturalpolymer selected from the group consisting of alginate, chitosan,hyaluronan derivatives and collagen, and combinations thereof.

Also optionally and most preferably, the biodegradable polymer isselected from the group consisting of poly caprolactone, poly-glycolicacid, poly-lactic acid, poly lactic co-glycolic, poly tartonic acid, orco-polymers thereof and combinations thereof.

Optionally, the 3-dimensional scaffold comprises a biodegradablematerial. Alternatively and optionally, the 3-dimensional scaffoldcomprises a non-biodegradable material.

Optionally, the 3-dimensional scaffold has a pore size in the range offrom about 50 microns to about 1000 microns.

Also optionally, the 3-dimensional scaffold features a coating layercomprising a material selected from the group consisting ofpoly-D-lysine, poly-L-lysine, collagen, fibronectin, ExtracellularMatrix (ECM) and hydrogel, or a combination thereof.

The above implant may optionally be used to repair at least a portion ofa bone lesion, which case the implant is optionally pre-shaped forinsertion into the bone lesion.

According to other embodiments of the present invention, there isprovided a method of producing a prosthetic implant, comprisingisolating, expanding and co-cultivating of at least two cell typesselected from the group consisting of mesanchymal stromal cells,osteoblasts, osteoclasts, chondrocytes and endothelial cells orprogenitors thereof on a 3-dimensional scaffold.

Optionally the cells are arranged in a three-dimensional manner on thescaffold for improved growth and/or viability. The cells are preferablygrown to high density. The cells are optionally grown to a density of atleast 10⁶ cells/ml, preferably grown to a density of at least 5×10⁶cells/ml and more preferably grown to a density of at least 10⁷cells/ml.

Also optionally the three-dimensional scaffold provides an environmentsupporting the growth of high density cell growth through the use ofimproved media flow.

Preferably the expansion is performed in a flow system allowing thegrowth of high density cultures, such as a bioreactor.

More preferably, the bioreactor comprises a growth matrix. Optionally,the growth matrix is in sheet form. Preferably, the sheet comprisesnon-woven fiber. Alternatively, the sheet comprises open-pore foamedpolymers. More preferably, a thickness of the sheet is from about 50 toabout 1000 microns. Most preferably, the sheet comprises pores having adiameter of from about 10 microns to about 100 microns. Optionally andmost preferably, the sheet comprises fibers having a diameter of fromabout 0.5 microns to about 200 microns. Optionally, the diameter is fromabout 10 microns to about 20 microns.

Optionally the 3-dimensional scaffold is pre-shaped for implant into abone lesion.

Optionally the mesenchymal cell isolation process comprises preparingsamples of bone marrow, fat tissue, mobilized peripheral blood, placentaor cord vein obtaining a cell suspension, centrifuging the suspension;and collecting precipitated mesenchymal stromal cells and mesenchymalcells.

Optionally the hematopoietic and endothelial cell isolation processcomprises preparing samples of bone marrow; fat tissue; peripheralblood; mobilized peripheral blood, cord blood, cord vein or placenta,obtaining a cell suspension, centrifuging the suspension; and collectingprecipitated cells.

Optionally the co-cultivation step comprises subjecting the cells to anosteogenic stimulus.

Optionally the osteogenic stimulus comprises contacting the cells with amolecule selected from the group consisting of dexamethasone, sodiumβ-glycerophosphate, 1,25 dihydroxycholecalciferol calcitriol, andL-ascorbic acid-2-phosphate (10-500 nM). Preferably, the osteogenicstimulus comprises exposure to shear forces.

Optionally a concentration of the dexamethasone is in the range of fromabout 10 to about 200 nM.

Optionally a concentration of the sodium β-glycerophosphate is in therange of from about 5 to about 25 mM.

Optionally, a concentration of the 1,25 dihydroxycholecalciferolcalcitriol is in the range of from about 5 to about 50 nM.

Optionally the co-cultivation step is carried out in a medium comprisingat least one of a growth factor and a cytokine.

Preferably the growth factor or cytokine is selected from the groupconsisting of transforming growth factor beta (TGF beta), insulin-likegrowth factor-1 and 2 (IGF-1 and 2), PDGF (platelet derived growthfactor), osteogenic protein-1 (OP-1), fibroblast growth factor (FGF),FGF-2, FGF-9, FGF-10, PTH (parathyroid hormone), PRP (platelet richplasma), EGF (epidermal growth factor), VEGF (vascular endothelialgrowth factor) and a Bone Morphogenic Protein (BMP) family member. Morepreferably, the bone morphogenic protein is selected from the groupconsisting of BMP-2, BMP-4 and BMP-7.

According to other embodiments, there is provided an implant constructedaccording to any method described herein.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying figures. Thedescription, together with the figures, makes apparent how embodimentsof the invention may be practiced to those skilled in the art. It isstressed that the particulars shown in the figures are by way of exampleand for purposes of illustrative discussion of embodiments of theinvention.

In the figures:

FIG. 1 illustrates a bioreactor system for growth of cells in accordancewith the principles of the present invention;

FIG. 2 shows the results of seeding BM-derived MSCs on the carrier inco-culture with hematopoietic progenitors cultured in 3-D dynamic flowsystem; demonstrate the cells 3-D co-culture interaction;

FIGS. 3 and 4 show Scanning Electron Microscope (SEM) and confocalmicrographs of 3-D synthetic scaffold seeded with MSCs, cultured indynamic flow system; demonstrate the cells density along and between thefibers;

FIG. 5 shows light microscopy image of MSC cultures, 7 days afterinducing differentiation to osteoblasts;

FIG. 6 shows MSC differentiation to osteoblasts, and EDS spectrumanalysis of elements in molar concentration in MSCs cultures after 3weeks;

FIG. 7 shows a microscopic view of a co-culture containing two celltypes (osteoprogenitor cells and endothelial cells) after 7 and 14 daysin culture;

FIG. 8 shows Alizarin red staining in co-cultures containing two celltypes (osteoprogenitor cells and endothelial cells); demonstrate thosecells 3-D interaction

FIG. 9 shows that positive immunostaining for osteocalcin was higher inco-cultures of osteoprogenitors and endothelial cells after 14 days inculture;

FIG. 10 shows a general view of micro sphere construct loaded withosteoprogenitor and endothelial cells before and after seeding; theco-culture of Osteoprogenitor cell (CFDA, green) and Endothelial cells(Hoecht, blue) cultured on microspheres construct; demonstrating theco-localization of both endothelial cells and osteoprogenitors seeded onthe micro sphere scaffold;

FIG. 11 shows the mesenchymal stromal cells ability to different invitro into chondrocytes;

FIG. 12 shows the in vivo critical size defect experimental model andthe ability to create a non healing bone defect;

FIGS. 13 and 14 show the preclinical results based on using the in vivoCritical size defect and demonstrating the 3-D multi cells cultureability to heal a non healing bone defect;

FIG. 15 show the preclinical results demonstrating the biodegradableartificial bone transplanted within this in vivo model to be accepted bythe recipient and to allow the development of complete organ includingblood vessels;

FIG. 16 demonstrates cell-scaffold constructs of GFP labeled MSCscultured in 3D scaffolds;

FIG. 17 is a front view of an external fixation system according to thepresent invention;

FIG. 18A illustrates a temporal positioning and fixation of a limb of amouse;

FIGS. 18B-21 schematically illustrate a process of a creation of the CSDin a femur of a small animal; and

FIGS. 22A and 22B illustrate other embodiments of the external fixationsystem according to the present invention.

DESCRIPTION OF EMBODIMENTS

The present invention, in at least some embodiments, provides aprosthetic implant comprising a biocompatible 3-dimensional scaffold andat least two cell types selected from the group consisting ofosteoblasts, osteoclasts, chondrocytes, and endothelial cells, orprogenitors thereof.

The use of at least two cell types is in sharp contrast with prior artthat depends upon expansion of only one type of progenitor cells—namely,the pre-osteoblasts, such as disclosed in U.S. Pat. No. 6,811,776.

The present invention also provides, in at least some embodiments, amethod of producing the prosthetic implant described above, the methodcomprising the steps of isolation, expansion and co-cultivation of atleast two types of cells onto a 3-dimensional scaffold.

The 3-D scaffolds are pre-fabricated to the required size and shape. Aplurality of cells and stem cell from different sources (MSC ormesenchymal stem cells together with HSC or hematopoietic stem cellsand/or endothelial cells) in pre-determined ratios are preferably firstcultivated and expanded separately. Next they are preferably cultivatedand co-expanded ex vivo under sterile conditions on the 3-D scaffold,using conventional culture medium, such as DMEM, RPMI, with supplementsof human serum (from autologous or allogeneic sources) or animal serum,or in serum-free media that allows the attachment and growth of adherentcells. Culture medium that supported the initial growth and expansionphase of these cells may optionally be replaced by another cell cultureformula that supports the differentiation of these cells and boneformation.

HSC are seeded onto the founding 3-D culture system, which is the 3Dscaffold containing the MSC and osteoblasts with or without endothelialcells and/or endothelial progenitors, in a fixed ratio. Alternatively,HSC are seeded simultaneously or substantially simultaneously with theMSC and osteoblasts, with or without endothelial cells. A preferredratio of non-HSC to HSC is between 5:1 to 5000:1, respectively. Morepreferentially, the ratio is between 20:1 to 300:1. According toexperimental evidence performed by one or more of the inventors, theseratios were shown to be preferred (data not shown). For the mereexpansion of the cells and stem cells, serum-free or serum-containingmedia are employed. During the expansion phase, serum-containing mediais preferably free of supplemented bioactive molecules of the followinggroups—hormones, growth factors, chemokines and cytokines.

To promote downstream differentiation of the expanded cells into boneforming cells needed in order to create the bone-like prosthetics,co-cultures grown as previously described may be exposed to osteogenicstimuli. These may include the mere presents of shear forces generatedin the flow-through bioreactor system and/or a culture differentiationmedium that contains, for example, one or more of the followingmolecules: dexamethasone, calcitriol (vitamin D derivative), sodiumβ-glycerophosphate and L-ascorbic acid-2-phosphate.

Optionally, the growth medium may be supplemented with growth factorsand cytokines, such as, for example, one or more of: transforming growthfactor beta (TGF beta), insulin-like growth factor-1 (IGF-1), osteogenicprotein-1 (0P-1), fibroblast growth factor (FGF) members like FGF-2,FGF-9 and FGF-10 and members of bone morphogenic proteins (BMP)especially BMP-2, BMP-4 and BMP-7.

Preferably, the entire implant is then transplanted into apre-determined site of bone loss.

Exemplary, illustrative non-limiting applications of such implantsand/or prostheses and/or procedures according to the present inventioninclude bone replacement, bone augmentation (for example in cases ofbone weakness, osteoporosis and/or other conditions in which bone matteris reduced but not necessarily missing), dental applications (which mayoptionally relate to replacement for bone loss and/or boneaugmentation), specific bodily areas of replacement such sinuses inwhich semi solid media or gel may optionally be used as scaffold ratherthan a rigid scaffold (which may then optionally spread to fill thearea), combined bone/cartilage applications (in which the scaffold mayoptionally combine both types of structures, which may optionally beseparated within the scaffold, for example for treatment of jointsincluding but not limited to knees, elbows, ankles etc), plastic surgeryapplications, bone or cartilage repair, cartilage replacement,orthopedic applications and other hard tissue applications, or cartilageapplications alone. Cartilage is optionally prepared from allogeneicmaterials.

The implants may optionally be prepared freshly for each applicationand/or may optionally be kept frozen in liquid nitrogen for examplebefore use. Also the implants may optionally be partially prepared andthen finished at the time of use, for example to permit the use ofautologous cells from the subject to be treated.

Cells are preferably expanded and co-cultivated in a dedicatedbioreactor system. The preliminary results presented in the Examplessection below demonstrating that a dynamic flow system, such as abioreactor for example, stimulates optimal cell density and cellviability in a 3-D construct, which is preferred for successful in-vivoimplantation.

FIG. 1 illustrates a sample for plug flow bioreactor system may be usefor the growth of cells in accordance with the principles of the presentinvention.

The bioreactor is described in detail in U.S. Pat. No. 6,911,201, whichis incorporated by reference as if fully set forth herein. In thispatent, a plug flow bioreactor system which allows the growth andprolonged maintenance of high density stromal cells cultures, thatclosely mimics the 3D bone marrow microenvironment. The cells wereseeded on porrosive (ie having pores) inorganic carriers made of a nonwoven fabric matrix of polyester, enabling the propagation of large cellnumbers in a relatively small volume. The structure and packing of thecarrier have a major impact on oxygen and nutrient transfer, as well ason local concentrations and released stromal cell products (e.g., ECMproteins, cytokines). In addition, the capacity of the mesenchymal andstromal cells cultured in this system to promote the maintenance andexpansion of transplantable human hemopoietic stem cells has beendetermined to be far superior over prior art methods.

The bioreactor comprises a medium reservoir 1; gas mixture container 2;gas filters 3; injection points 4; 5 plugs or containers of varioussized plugs containing a pre-shaped 3-D scaffold; flow monitors 6; flowvalves 6 a; conditioned medium collecting and separating container 7;container for medium exchange 8; peristaltic pump 9; sampling point 10;container for medium exchange 11; monitor 12; steering device 14; and pHprobe.

Preferably a continuous flow system is used. However, it should beunderstood that any controlled flow system could be used in place of thebioreactor of FIG. 1, which is intended as a non-limiting, illustrativeexample only. Flow, adequate but not excessive, levels of dissolvedoxygen, control of pH, glucose level and temperature are the mostimportant components of such a system. For example, the bioreactor mayoptionally be implemented according to the P1uriX™ Bioreactor(Pluristem, Haifa, Israel), New Brunswick bioreactors and controlledstir tanks or rolling bottles system.

The flow system or bioreactor described herein includes the preferredfeatures of supporting 3-D MSCs cultures with a continuous flow system.The plug-flow bioreactor described herein is capable of supporting thelong-term growth of primary human MSC, osteoblasts, endothelial and HSC3-D multi cells-cultures.

The use of 3-D multi cells-cultures in the bioreactor is not onlyessential for the establishment of superior cell-cell contact (viaunique “niches” and cell-cell, cell-ECM interactions), but also for theproduction of known and novel soluble and membrane-bound cytokines. The3-D multi cell culture can facilitate the supplementation of suchbioreactors with appropriate cytokines, by using genetically engineeredcytokine-producing variants.

In sharp distinction to background art methods, the bioreactor of thepresent invention employs a growth matrix that substantially increasesthe available attachment surface for the adherence of the MSCs,osteoblasts, endothelial and HSCs so as to mimic the mechanicalinfrastructure of bone microenvironment and allows in vitro boneformation. The growth matrix comprises a porous material as described ingreater detail below. For example, for a growth matrix of 0.5 mm inheight, the increase is by a factor of at least from 5 to 30 times,calculated by projection onto a base of the growth matrix. Such anincrease by a factor of about 5 to 30 times, is per unit layer, and if aplurality of such layers, either stacked or separated by spacers or thelike, is used, the factor of 5 to 30 times applies per each suchstructure.

When the matrix is used in sheet form, preferably non-woven fibersheets, or sheets of open-pore foamed polymers, the preferred thicknessof the sheet is about 50 to 1000 μm or more, there being providedadequate porosity for cell entrance, entrance of nutrients and forremoval of waste products from the growing tissues.

According to a preferred embodiment the pores have an effective diameterof 10 μm to 100 m. Such sheets can be prepared from fibers of variousthicknesses, the preferred fiber thickness or fiber diameter range beingfrom about 0.5 μm to 20 μm, still more preferred fibers are in the rangeof 10 μm to 15 μm in diameter.

The matrix sheets may also be cut, punched, or shredded to provideparticles with projected area of the order of about 0.2 mm² to about 10mm², with the same order of thickness (about 50 to 1000 μm).

The structures of the 3D scaffolds according to some embodiments of thepresent invention may be supported by, or bonded to, a porous supportsheet or screen providing for dimensional stability and physicalstrength.

Thus a 3-D scaffold in concert with a dynamic flow bioreactor asillustrated in FIG. 1 provides for long-term cell viability of in vitrocultured cells seeded in 3D scaffolds which are intended for in vivoapplication.

As shown in FIGS. 2-4, this growth system provides the necessarycritical mass cell/volume and physiological milieu, including a constantsupply of essential oxygen and nutrients, thus closely mimicking the invivo conditions.

FIGS. 3 and 4 show Scanning electron microscope (SEM) and confocalmicrographs of 3-D synthetic scaffold seeded with human MSCs cultured indynamic flow system; demonstrate the cells density along and between thefibers.

The mesenchymal cells, osteoblasts, chondrocytes, endothelial and HSCthat are used in various embodiments of the present invention are ofautologous, syngeneic or allogeneic sources. For transplantation ofhematopoietic or endothelial cells from allogeneic donor sources, HLAtyping is performed and only sufficiently matched cells are being used.The most common cause of bone graft failure is graft rejection (Hoffmannet al, 1998). However, Mesenchymal cells and MSCs, express low levels ofMHC antigens (Sun et al, 2003; Niemeyer et al, 2004) and MSC were shownto inhibit T-cell responses (Krampera et al, 2003; Maitra et al, 2004).Transplanted allogeneic Mesenchymal cells and MSCs could be detected inrecipients at extended time points, indicating lack of immunerecognition and clearance (Aggarwal and Pittenger, 2005). Accordingly,MSCs and Mesenchymal cells like osteoblasts and chondrocytes may begenerated from autologous or allogeneic sources. Endothelial cellsthough expressing only low levels of HLA antigens (Neppert et al, 1984;Shahgasempour, 1998; Johnson, 2000) are immunogenic and could mediatevascular rejection (endothelialitis). This also applies for cells of theHSCs origin like osteoclasts. As such, according to the presentinvention and prior to the buildup of bone prosthetics from allogeneicsources, HLA repertoire on donor cells should be typed. Only closedmatching allogeneic endothelial cells, HSCs and osteoclasts should bemanipulated.

The 3-dimensional scaffold of the present invention comprises anon-toxic and biocompatible, biodegradable or non-biodegradablematerial, which may be prepared in three dimensions structure in orderto support the growth of 3-dimensional cell cultures and promote guidedtissue generation.

The scaffold preferably comprises a biocompatible polymer having poresize in the range of from about 50 to about 2000 microns, more in therange of from about 250 to about 750 microns. The physical environmentsthat support the growth of the co-cultures on the said 3-D matrixes maybe chosen from static systems involving cell culture grade flasks anddishes. However, culture of cells in scaffolds sufficiently large tobridge critical-sized defects is problematic. Simple diffusion may beunable to provide sufficient nutrients deep into large scaffolds.Consequently, under static culture conditions cells may preferentiallyproliferate at the scaffold periphery only. Under the growth conditionsof the present invention, various alternative machineries couldoptionally and preferably be used to allow media flow and convey theculture media uniformly. These include but are not limited to stirredtanks, spinner flasks, rotary vessels, rolling bottles, rolling basketsand a perfusion plug flow or flow-through (plug flow) system (seeexample 1, FIG. 1 for details).

The 3-D scaffold of the present invention material can be powdered, semisolid or gel-like, so that it is suitable for use in implants that haveno mechanical strength function requirements. However, the scaffold canalso be substantially preshaped and sponge-like or porous and can e.g.comprise an organic, ceramic or metallic material and, as a function ofthe specific chosen carrier material, can fulfill a mechanical strengthfunction and allow high density cells growth. The preshaped implant mayalso optionally be prepared by cutting or otherwise altering thestructure of the 3-D scaffold to be suitable for the characteristics andrequirements of the desired implant.

The 3-D scaffold of the present invention may optionally comprise dry,frozen or dematerialized bone particles or polymer selected from thegroup consisting of aliphatic polyesters, poly (amino acids), co-poly(ether-esters), polyalkylenes oxalates, polyamides, tyrosine derivedpolycarbonates, poly (iminocarbonates), polyorthoesters, polyoxaesters,polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, bioactive glass and calcium phosphatederivatives. Biodegradable polymers may also be used comprising naturalbiopolymers like alginate, chitosan, fibrin, fibronectin, hyaluronanderivatives and collagen. Biodegradable synthetic polymers may includederivatives such as poly caprolactone, poly-glycolic acid, poly-lacticacid and poly lactic co-glycolic and poly tartonic acid.

The scaffold is preferably porous, having pore size of between 50microns and 2000 microns. This range was found to be suitable accordingto experiments performed by one or more of the inventors, such thatpores which are smaller than 50 microns do not permit the cells enter;pores larger than 2000 microns do not provide sufficient support for the3D scaffold.

The implants need to have mechanical strength and require a more rigidstructure which would presumably not be injectable. The implant mayoptionally be pre-shaped to the precise tissue gap size and structureand may be ready for transplantation into the lesion to be repaired.Optionally, the implant may be injectable and serve as a filler. Asfiller, the 3-B cultures may optionally and preferably be in aninjectable form, such as a gel, semisolid or powder for example.

Exemplary embodiments of the invention are discussed herein below withreference to specific materials, methods and examples. The material,methods and examples discussed herein are illustrative and not intendedto be limiting. In some embodiments, methods and materials similar orequivalent to those described herein are used in the practice or testingof embodiments of the invention. It is to be understood that theinvention is not necessarily limited in its application to the detailsof construction and the arrangement of the components and/or methods setforth in the following description and/or illustrated in the drawings.The invention is capable of other embodiments or of being practiced orcarried out in various ways.

EXAMPLES

Materials and Methods

Bioreactor: The bioreactor used in accordance with the teachings of thepresent invention was constructed in accordance with the design shown inFIG. 1, and described in detail above.

The glassware was designed and manufactured by the inventors andconnected by silicone tubing (Degania, Israel).

The carriers for the cells were rotated overnight in phosphate bufferedsaline (PBS; Beit Ha'Emek Industries, Israel) without Ca²⁺ and Mg²⁺,followed by removal of the PBS and released debris.

Each column was loaded with 10 ml packed carrier. The bioreactor wasfilled with PBS without Ca²⁺ and Mg²⁺, all outlets were sealed and thesystem was autoclaved (120° C., 30 minutes). The PBS was removed viacontainer [8] and the bioreactor was circulated in a 37° C. incubatorwith 300 ml Dulbecco's high-glucose medium (DMEM; GIBCO BRL) containing10% heat-inactivated fetal calf serum (FCS; Beit Ha'Emek Industries,Israel) and a Pen-Strep-Nystatin mixture (100 U/ml:100 μg/ml:1.25 μn/ml;Beit Ha'Emek), for a period of 48 hours. Circulating medium was replacedwith fresh DMEM containing the above +2 mM L-glutamine (Beit Ha'Emek).

MSCs/Stromal cells: Primary human marrow MSC and stromal cultures wereestablished from aspirated stromal marrow of hematological healthydonors undergoing open-heart surgery. Alternatively MSC and stromalcultures were established from placenta or adipose tissues.

Bone marrow derived stromal cells—, marrow aspirates were 3-fold dilutedin Hank's Balanced Salts Solution (HBSS; GIBCO BRL) and were subjectedto Ficoll-Hypaque (Robbins Scientific Corp. Sunnyvale, Calif.) densitygradient centrifugation. Marrow mononuclear cells (<1.077 gm/cm³) werecollected, washed 3 times in HBSS and resuspended in long-term culture(LTC) medium, consisting of DMEM (Beit Ha'Emek) supplemented with 12.5%FCS, 12.5% horse serum (Beit Ha'Emek), 10⁻⁴ M β-mercaptoethanol (Merck)and 10⁻⁶ mol/L hydrocortasone sodium succinate (Sigma). Cells wereincubated in 25 ml tissue culture flasks (Corning) for 3 days at 37° C.(5% CO2) and then for 3 days at 33° C. (5% CO2) with weekly culturerefeeding. Stromal cells from individual donors were employed for eachbioreactor. Primary stromal cell cultures were split by trypsinization(0.25% Trypsin and EDTA in Puck's Saline A; Beit Ha'Emek) every 10 days,to allow sufficient cell expansion. Cultures were maintained at 33°C.-37° C. in LTC medium.

Placenta derived stromal cells—Inner parts of a full-term deliveryplacenta (Bnei Zion medical center, Haifa, Israel) were cut understerile conditions, washed 3 times with Hank's Buffer and incubated for3 h at 37° C. with 0.1% Collagenase (1 mg/ml tissue; Sigma-Aldrich, St.Lewis, Mo.). Using gentle pipeting, suspended cells were then washedwith DMEM supplemented with 10% FCS, Pen-Strep-Nystatin mixture (100U/ml:100 ug/ml:1.25 un/ml) and 2 mM L-glutamine, seeded in 75 cm² flasksand incubated at 37° C. in a tissue culture incubator under humidifiedcondition with 5% CO₂ Thereafter, cells were allowed to adhere to aplastic surface for 72 hours after which the media was changed every 3-4days. When reaching 60-80% confluence (usually 10-12 days), cells weredetached from the growth flask using 0.25% trypsin-EDTA and seeded intonew flasks. Cultured cells were thereafter collected for analysis or forculturing in bioreactors.

Adipose derived stromal cells—Stromal cells were obtained from humanadipose tissue of liposuction procedures (Rambam Haifa, Israel). Adiposetissue was washed extensively with equal volumes of PBS and digested at37° C. for 30 min with collagenase (20 mg/ml). Cells were then washedwith DMEM containing 10% FCS, Pen-Strep-Nystatin mixture (100 U/ml:100ug/ml:1.25 un/ml) and L-Glutamin and centrifuged at 1200 rpm for 10 minRT, resuspended with lysing solution (1:10; Biological Industries, BeitHa'emek, Israel, in order to discard red-blood cells) centrifuged andresuspended with DMEM containing 10% FCS, Pen-Strep-Nystatin mixture(100 U/ml:100 ug/ml:1.25 un/ml) and L-Glutamin. Washed cells were thenseeded in a sterile tissue culture medium flask at 3-10*10⁷ cells/flask.At the next day cells were washed with PBS to remove residual RBC anddead cells. The cells were kept at 37° C. in a tissue culture incubatorunder humidified condition with 5% CO₂ The medium was changed every 3 to4 days. At 60-80% confluence, the cells were detached from the growthflask using 0.25% trypsin-EDTA (Beit Ha'Emek) and seeded into newflasks.

Seeding of MSCs onto the 3-D cultures: 3-5 week MSCs were trypsinizedand the cells washed 3 times in HBSS, resuspended in the bioreactormedium (see above), counted and seeded at 10⁶ cells/ml in 10 ml volumesvia an injection point ([4], FIG. 1) onto 10-100 ml 3-D carriers in theglass column of the bioreactor. Immediately following seeding,circulation was stopped for 0.25-24 hours to allow the cells to settleon the carriers. The cell growth in the bioreactor was monitored byanalyzing the glucose demand of the cultures and by removal of carriersand cell enumeration by the MTT method (56). When the cells cultureswere confluent, medium was replaced with LTC medium.

Isolation of Hematopoietic CD34+ cells: Umbilical cord blood (CB), BMand peripheral blood samples were taken under sterile conditions andfractionated on Ficoll-Hypaque and buoyant (<1.077 gr/cm³) mononuclearcells collected. The Cells were incubated with anti-CD34 antibodies andisolated by midi MACS (Milteny Biotech).

Isolation of endothelial cells: Endothelial culture cells were collectedfrom peripheral blood. Buffy coat mononuclear cells from 50 or 100 ml ofblood were resuspended in EGM-2 medium (Clonetics Inc, USA) withoutfurther cell subpopulation enrichment procedures and placed into platescoated with type I collagen (Becton Dickinson, USA). The plate wasincubated at 37° C. in a humidified environment with 5% CO2. Culturemedium was changed daily. After 24 hours, unattached cells and debriswere removed by washing with medium. This procedure leaves the attachedendothelial cells, as identified by morphology and staining withanti-endothelial monoclonal antibody P1H12, plus other mononuclear cellsthat died out within the first 2-3 weeks of culture.

The cells were kept at 37° C. in a tissue culture incubator underhumidified condition with 5% CO₂ The medium was changed every 3 to 4days. At 60-80% confluence, the cells were detached from the growthflask using 0.25% trypsin-EDTA and seeded into new flasks coated with 50μg/mL of fibronectin (Sigma) as substrate.

MSCs-HSCs co-cultures: Isolated, CB derived CD34+ cells were seeded atequivalent numbers (about 5×10⁵) onto monolayer or bioreactor containingequivalent densities of confluent MSCs. Upon addition to the bioreactor,medium flow was stopped for 0.25-16 hours to enable contact with MSCsand was re-initiated at a rate of 0.1-1.0 ml per minute. CD34+ cellseeded-MSCs carriers were removed for control studies in the absence ofmedium exchange. Co-cultures were maintained in growth medium, with orwithout cytokines. At various times (up to 4 weeks), nonadherent cellswere collected from monolayer supernatants or from circulating culturemedium via a container ([8], FIG. 1). Adherent cells were collected viasequential trypsinization and exposure to EDTA-based dissociation buffer(GIBCO BRL), followed by gentle pipetting of the cells. To avoid thepresence of MSCs in the resulting suspension, the cells were resuspendedin HBSS+10% FCS and were subjected to a 60 minutes adhesion procedure inplastic tissue culture dishes (Corning), at 37° C.

Osteogenic culture differentiation: Final bone tissue formation isexecuted in osteogenic culture differentiation medium composed of one ormore of the following molecules in preferred concentration:dexamethasone (10-200 nM) (Sigma), sodium β-glycerophosphate (5-25 mM)(Sigma), 1,25 dihydroxycholecalciferol (calcitriol: 5-50 nM) (Sigma) andL-ascorbic acid-2-phosphate (10-500 nM) (Sigma).

Chondrocyte differentiation: Mesenchymal cells were seeded inconcentration of 2*105 cells/tube—cells suspended in lml medium withchondtogenic cocktail: DMEM HG, BMP-6 [500 ng/ml] (Sigma), TGF-b3 [10ng/ml] (Sigma), ITS+premix [dil. :20 of the dil. Stock(:100)] (Sigma),Dexamethasone [100 nM] (Sigma), L-ascorbic acid 2-phosphate [50 mg/ml](Sigma), Sodium pyruvate [100 mg/ml] (Sigma), Proline [40 mg/ml](Sigma), Pen/Strep/Nys 1% , Glutamine 1% . The medium should be replacedevery 2 days, for 21 days. After 21 days of differentiation, the culturewas stained with Alcian blue (Sigma). Staining procedure:Fixation—formalin fixed, paraffin embedded tissue sections.Deparaffinize slides and hydrate to distilled water. Stain in alcianblue solution for 30 minutes. Wash in water. Counterstain in nuclearfast red solution for 5 minutes. Dehydrate through 95% alcohol, 2changes of absolute alcohol, 3 minutes each.

Example 1 Growth of MSC and Osteoblasts 3-D Cultures in Flow System

Example 1 describes typical expansion experiments for co-culturing ofthese components, but with addition of the components at separate times.

In one experiment 30000 BM-derived MSCs were seeded on each carrier andthe system was cultivated for 50 days. Results are shown in FIG. 2A.During the first six weeks, no cell expansion was noted but thefollowing week was characterized by extensive cell proliferation. At theend of the 50-day period, expansion was measured at 2.8 folds (total of85000 cells/carrier). 10000 CB derived CD34+ cells were then seeded ontothe system of which early HSC (CD34+38-45+) counted for only 2500 cells(ratio of MSCs to HSC at time of co-culture establishment). Fourteendays later, at the time of harvest, MSCs cells were shown to expand byadditional factor of 2. During the same period of time, HSC number inthis system was increased by 42 folds.

In the second experiment, 42000 BM-derived MSCs were seeded on eachcarrier. Results are shown in FIG. 2B. An initial drop of stroma countwas seen, that persisted for 24 days. 9400 CD34+ cells were then seededon each carrier and culture allowed proceeding for additional 13 days.At the end of this period there was a net increase of 20% in the numberof MSCs whereas HSC expanded by 90%.

These results show that by varying the initial expansion period ofstroma mono-culture and the ratio of seeded CD34+ to MSCs at time ofestablishment of the co-culture system, a control over final cell blendcould be attained. This is a desired ability where demands for strengthand elasticity of bone material could vary.

FIG. 3 shows Scanning Electron Microscope (SEM) and confocal micrographsof 3-D synthetic scaffold seeded with MSCs in cultured in static and indynamic flow system:

(A) SEM micrograph of human MSCs seeded on 3D scaffold;

(B) Confocal image of human MSCs seeded on 3D scaffold. Numerouselongated cells are seen throughout the scaffold. Note auto fluorescenceof the scaffold fibers (green: Confocal image of static culture ×10).

(C) SEM of scaffold cultured with human MSCs in dynamic flow growthsystem. Note, dense cell distribution and extra cellular matrix as wellas distinct 3-D meshed structure of the scaffold containing cells whichare aligned along and between the fibers.

(D) Confocal microscope projection image of human MSCs cultured on 3-Dscaffold construct in dynamic flow system. Numerous elongated cells areseen throughout the scaffold. Note cell cytoplasm (blue) and nuclei(red).

FIG. 4 shows light microscopy images of the scaffold containingmesenchymal cells after in vivo subcutaneous implanting in nude mouse:

(A) The implant appears to be fully integrated with the surroundingtissues. Dense cell distribution and matrix containing infiltrated bloodvessels (arrows) are observed.

(B) A higher magnification of the previous figure showing cellsattachment to the nanofibers and blood vessels containing erythrocyteswithin the scaffold (arrows).

Example 2 MSC Differentiation to Osteoblasts

This Example relates to 2D and 3D cultures of MSC cells which are thendifferentiated into preosteoblasts and osteoblasts. The 3D culture issimilar to Example 1, except that the component cells are placedtogether simultaneously in culture.

Final bone tissue formation is executed in osteogenic culturedifferentiation medium composed of one or more of the followingmolecules in preferred concentration: dexamethasone (10-200 nM) (Sigma),sodium β-glycerophosphate(5-25 mM) (Sigma), 1,25dihydroxycholecalciferol (calcitriol: 5-50 nM) (Sigma) and L-ascorbicacid-2-phosphate (10-500 uM) (Sigma).

FIG. 5 shows light microscopy image of MSC cultures, 7 days afterinducing differentiation to osteoblasts.

FIG. 6 shows MSC differentiation to preosteoblasts and osteoblasts, andEDS spectrum analysis of elements in molar concentration in MSCscultures after 3 growth weeks.

Example 3 Co-Culture of Osteoprogenitor Cells and Endothelial Cells

Osteoprogenitor cells (OS) and Endothelial cells (C) were co-cultured ona 3-D scaffold for inducing osteogenesis in vitro.

Bone marrow MSCs—derived osteoprogenitor cells (OS) (50,000 cells/wellof 24 wells culture plate) were either plated alone or co-cultured withEndothelial cells (EC) (2,000 cells/well of 24 wells culture plate), on3-D hydrogel scaffold in {acute over (α)}-MEM medium containingosteogenic supplements, for up to 21 days. Endothelial cells weresimilarly cultured alone.

The effect of endothelial cells on the differentiation pattern ofosteoprogenitor cells to the osteogenic lineage was followed by AlizarinRed S, staining for demonstration of calcium deposits and by osteocalcininmmunostaining for demonstrating the synthesis of bone specificmacromolecules.

The results in FIG. 7-9 demonstrate the difference in proliferativeactivity of these two cell types. Thus when cultured alone, theosteoprogenitor cells proliferated at a lower rate than the endothelialcells. In addition, those results prove that the osteogenicdifferentiation progressed only when a high number of cells (close toconfluence) were present in the culture system. Thus, as compare to theendothelial cells, a higher number of osteoprogenitors was needed forthe culture.

In order to be able to co-culture these two cell types together, twoapproaches were used:

A: The osteoprogenitor cells were cultured from the beginning at alarger numbers (50,000 cells/group) and the endothelial cells wereplated at lower numbers (2,000 cells/group).

B: The osteoprogenitor cells were seeded first and only after 7 days(when their numbers increased) the endothelial cells were added to theculture. This approach was crucial in the 3-D micro sphere dynamicculture technique, since the osteoprogenitor cells formed bridgesbetween the micro spheres and caused their coalescence forming a clot,prior to the seeding of the endothelial cells (for additional week)

FIG. 7 shows a microscopic view of a 3-D co-culture containing both celltypes (osteoprogenitor cells and endothelial cells) after 7 and 14 daysin culture demonstrating that cell quantity increased in the culturefrom 7 to 14 days.

Osteoprogenitor cells and endothelial cells were co-cultured for 3, 7,10, 14 days on 3-D hydrogel scaffold, stained with Alizarin Red Sstaining, for calcium deposits.

As shown in FIG. 8, the combination of osteoprogenitor cells andendothelial cells caused induction of Alizarin Red S staining incultures containing both cell types after 14 and 21 days, as compared tocontrol cultures with osteoprogenitor cells only. This result indicatesinduced calcium deposition in the 3-D co-culture combination. Notegradual increase of alizarin red staining in co-culture after 10 and 14days. Osteoprogenitor cells and endothelial cells were co-cultured for 7and 14 days on hydrogel scaffold.

FIG. 9 shows that positive immunostaining for osteocalcin was higher inco-cultures of osteoprogenitors and endothelial cells after 14 days inculture, which indicates synthesis of bone-specific macromolecule. Notestaining after 14 days in culture (indicated by arrows)

The results shown in FIGS. 8 and 9 indicate that the 3-D combination ofthe two cell types enhanced calcium deposition, indicating enhancedosteogenic differentiation including mineralization, by alizarin red S,and promoted synthesis of bone specific, osteocalcin positivemacromolecules.

Example 4 Growing 3-D Co-Culture of Osteoprogenitor Cells andEndothelial Cells

Osteoprogenitor cells (OS) and Endothelial cells (C) were co-cultured on3-D hydrogel scaffold for inducing osteogenesis in vitro.

Bone marrow MSCs derived osteoprogenitor cells (OS) were plated inco-cultured with Endothelial cells (EC), on 3-D scaffold in {acute over(α)}-MEM medium containing osteogenic supplements, for up to 21 days.

Cell viability was followed by live/dead fluorescent markers. Theosteoprogenitor cells were pre labeled with CFDA (green fluorescence),and the endothelial cells were pre labeled with Hoecht (bluefluorescence). Osteoprogenitor cells (50,000 cells/construct) were firstseeded on micro sphere hydrogel scaffold in dynamic culture for up to 7days (FIG. 10), followed by seeding the endothelial cells (2,000cells/construct) for additional 7 days.

Cells cultured on the 3-D hydrogel scaffold were stained with a celltracer reagent: Carboxyfluorescein Diacetate Succinimidyl Ester (CFDASE). The non-fluorescent CFDA SE diffuses into the cells and uponcleavage by intracellular esterases become fluorescent indicating viableproliferating cells. Hoecht stain penetrates to the cell nuclei andenables detection of viable cells in the culture.

FIG. 10 demonstrate a general view of the 3-D micro spheres constructloaded with osteoprogenitor and endothelial cells. The two cellpopulations, the osteoprogenitors and the endothelial cells, were viableon the scaffold and can be used for the in vivo implants. The scaffoldmicrospheres were first seeded with osteoprogenitor cells (pre-labeledwith CFDA, green) and than were seeded with ECS (pre-labeled withHoecht, Blue). FIG. 10 also shows the interaction between the co-cultureof Osteoprogenitor cell (CFDA, green) and Endothelial cells (Hoecht,blue) cultured on the microspheres construct (1 week; dynamic culture)demonstrating the high density of viable osteoprogenitor and endothelialcells and the co-localization of both endothelial cells andosteoprogenitors seeded on the on the micro sphere scaffold, (overlap ofthe green and blue colors).

Example 5 Growing 3-D Co-Culture of Osteoblasts and Chondrocytes

One of the first needs for bone cartilage grafts is injuries to thecartilage of the knee joint. Injuries to the knee are not uncommon amongboth professional athletes as well as active non-professional sportsenthusiasts; and incidents of knee trauma are also among the unfortunateresults caused by accidents of all sorts. There is a large group ofpatients in need of a solution to damaged knee cartilage.

Allograft transplantation is a conventional method for treating suchinjuries. The advantages of allograft transplantation include goodresults for a longer time period post surgery and the fact thistreatment can be applied to a wider spectrum of patients. However, thelack of donors and the unavailability of these grafts, the necessaryviability of the chondrocytes and osteoblasts, as well as the safetyissue regarding the supplied donor tissue and the need for matching thesite-specific requirements (size, shape, cartilage thickness), limit theuse of those tissues.

Using bone cartilage replacement prosthetics and growing cellular-basedbone cartilage implants in vitro may allow the creation ofosteoarticular grafts, which comprise implant consisting of hyalinecartilage attached to bone. This complex graft can be grown in vitro,kept frozen and stored for indefinite periods of time. The cartilagebone graft received following thawing contains a high level of viablechondrocytes. This approach will allow the growth and supply of match,safe and available tissue. The ability to grow and cryopreservecartilage grafts also provides a way to store the in vitro createdtissues for long term, thereby creating a bank of various shapes, sizesand other specific characteristics including donor age, cartilagethickness, etc. Cellular-based bone cartilage implants may supply safertissue for transplantation, reduce the waiting time for an appropriatedonor and allow better matching of tissue shape and size for betterrepair success.

In order to achieve this goal, 3-D osteochondral allograft plugs, madeof osteogenic implants are treated on one side to induce the growth ofcartilage tissue. The final growth phase on the side of the plug isstimulated toward chondrocytes differentiation using growth factor suchas, but not limited to, TGF-beta (Sigma) BMPs (Sigma), retinoids(Sigma), FGFs (Sigma), GH (Sigma), IGFs (Sigma) and transferrin (Sigma).Following the cartilage growth, the allograft plugs are processed,cryopreserved and store ready for use. When needed, the plugs are thawedin the operating theatre prior to transplantation and transplanted usinga press fit technique.

FIG. 11 demonstrates the ability of ex vivo expanded 3-D mesenchymalcells to differentiate in vitro into mature chondrocytes.

Example 6 Transplanting 3-D Co-Culture of Osteoprogenitor Cells andEndothelial Cells

In order to further increase the osteogenic potential of scaffold-basedimplants, a cell-therapy approach was used to incorporateosteoprogenitor cell-derived from bone marrow Mesenchymal Stromal cells(MSCs) into the scaffold to enhance bone repair. Osteoprogenitor cells(OS) and Endothelial cells (EC) were co-cultured on the 3-D scaffolds invitro. The results presented in FIGS. 12-16 were validated by the use ofspecific osteogenic markers demonstrating that cultures of sufficientnumbers of osteogenic cells, endothelial cells and growth factors couldconceivably be used with scaffolds for bone tissue engineering torepairing bone loss in aging and in bone transplantation (FIG. 15).

The present inventors have demonstrated that the methods used for the invitro selection of the osteogenic subpopulation from MSCs cultures andthe methods used to incorporate them in scaffold are crucial forsuccessful transplants for future use in tissue engineering bone repair.The present inventors have also demonstrated that the scaffold ispreferably biocompatible for selected osteogenic cells and providessupport for proliferation and differentiation.

The scaffold is optionally and preferably biocompatible,osteoconductive, biodegradable and osteoinductive, but notimmunoreactive. The 3-D scaffolds provide the necessary support forcells to proliferate and maintain their capacity to differentiate.

Transition from a 2-D culture system to the 3-D scaffold provides asystem that imitates the natural 3-D structure of the body tissues andspecifically the structure of bone. The 3-D scaffolds containingMSC-derived osteoprogenitors and additional supporting cells can beemployed within transplants in order to enhance bone repair. The complexconstruct is intended to mimic the native in vivo microenvironment andthis necessitates construction of bioactive scaffolds which are alsocapable of supporting vascularization as well as cell proliferation andosteogenic differentiation.

Preclinical animal tests developed by the present inventors are acrucial step prior to conduction of the actual clinical trials andfinalized the preclinical tests aimed to validate the functionality ofthe transplanted cells, its safety parameters and the assessment ofnon-immunoreactivity of either the cells or the scaffold in the designedtransplanted cell-scaffold constructs. The described in vivo animaltests constitute a step midway between the in vitro studies and thehuman clinical applications, and are crucial for demonstrating thefunctionality of the designed cell-scaffold constructs.

FIG. 12 demonstrates the small animal model developed and used by thepresent inventors in the small animal (mice and rats) preclinicalstudies. The available methods include the use of an external fixationdevice for conducting a critical size defect (CSD) in long bone thatwill not heal. As FIG. 12 demonstrates, following the creation ofcritical defect and bone external fixation, the bone defect does notheal for more than 3 months.

FIGS. 13-14 demonstrate other critical gap animal models used by thepresent inventors for studying bone repair, including bone segmentaldefects conducted in rat tibia and the three-wall bone defect conductedin rat mandible. Both models tested by the present inventors havedemonstrated successful and complete bone repair within 6 weeksfollowing the new in vitro developed bone transplant.

FIGS. 15-16 demonstrate the use of immunodeficient mice strains (Nude,SCID/NOD) for testing human cells transplanted. The mice were treated bybone implant transplants (orthoptopic animal model). FIG. 15demonstrates full repair of the defect as shown by x-ray, histology andμCT. FIG. 16 demonstrates cell-scaffold constructs of GFP labeled MSCscultured in 3D scaffolds and transplanted subcutaneously/intramuscularly(ectopic animal models), revealing new bone formation and the formationof blood vessels in the transplant animal.

Example 7 Critical Gap Bone Repair Model Test In Vivo

The present invention, in at least some embodiments, is generallyconcerned with creation of critical size defects (CSD) in bones ofanimals, including humans, using external bone fixators.

In accordance with one aspect of the present invention, there isprovided an external fixation system for creating a CSD in a long boneof an animal, the system comprising a frame composed of at least twopins for percutaneous insertion from the lateral to the medial side ofsaid bone spaced apart in a distance greater than the length of the CSD;and at least one moldable bridge for fixation of said pins with respectto each other and over said bone in a manner to prevent the pins fromany longitudinal or rotational displacement with respect to said boneand with respect to each other.

The pins are inserted perpendicular or with a certain angle with respectto the long axis of the bone and are adapted to be long enough toprotrude beyond the opposite cortices thereof. In this case two moldablebridges are used for fixation at both the lateral and the medial sidesof the bone.

Any one or more of the following features may be included in the systemaccording to the present invention. For example, the system may furthercomprise 4 pins, two of them positioned at the upper third of the bone,and the other two are positioned at the lower third of the bone, havinga CSD there between. In this case the system may further comprise morethan one bridge, each bridge fixating one couple of pins (one pin fromthe upper third of the bone and another pin from the lower third of thebone).

Each couple of pins may be inserted perpendicular or with a certainangle with respect to the long axis of the bone. The system may compriseany even or odd number of pins. The bridge may be made of fast hardeningacrylic materials, such as dental acrylic paste. The system is suitablefor small animals such as mice, as well for larger animals such ashorses, cows, bulls and other agriculturally important animals, or evenhumans.

According to a further aspect of the present invention, there isprovided a kit, the kit comprising the external fixation system forcreation of a CSD in a long bone of an animal, the system comprising aframe composed of at least two pins for percutaneous insertion from thelateral to the medial side of said bone spaced apart in a distancegreater than the length of the CSD; and at least one moldable bridge forfixation of said pins with respect to each other and over said bone in amanner to prevent the pins from any longitudinal or rotationaldisplacement with respect to said bone and with respect to each other;and a mold made of flexible material for temporarily positioning andfixation of a limb of the animal to allow a desired position thereof.The mold is soft and may be made of semi-rigid silicon.

According to a further aspect of the present invention, there isprovided a method for creation of a critical size defect (CSD) in a longbone of an animal, the method comprising: providing an external fixationsystem comprising a frame composed of at least two pins and a at leastone moldable bridge; temporarily positioning and fixation of a limb witha flexible mold of the animal to allow a desired position thereof, inwhich there is no damage occurs to the blood vessels and nerves of theanimal's limb; percutaneous insertion of said pins from the lateral tothe medial side of said bone in a distance greater than the length ofthe CSD; fixation of said pins with said moldable bridge with respect toeach other and over said bone in a manner to prevent the pins from anylongitudinal or rotational displacement with respect to said bone; andinduction of the CSD.

In case that two moldable bridges are used for fixation at both thelateral and the medial sides of the bone, as described above, the moldis removed before the fixation of the pins at the medial side of thebone.

In addition to creating a CSD, the method according to the presentinvention may be used for creating of fractures, partial fractures andimmobilizations of the animal bones and joints (between femur andtibia).

Although the below detailed example centers around mice as a test modelanimal, in fact the system and method may be adapted for any largeranimal by one of ordinary skill in the art.

FIG. 17 illustrates an external fixation system generally designated 110for creating a critical size defect (CSD) 130 in a long bone 150 of ananimal (not shown), having a lateral side 140 and a medial side 160. Thesystem is designated for use with small animals, but is adapted to beused with large animals as well. The system comprising a first couple ofpins 170 and a second couple of pins 190, protruding from both thelateral 140 and the medial 160 sides of the bone 150, positioned inparallel relation at the right and the left sides of the CSD 130. Thefirst and the second couples of pins 170 and 190 are spaced apart at adistance greater than the length of the CSD. The pins 170 and 190 arefixated over the bone 150 and with respect to each other with a lateralmoldable bridge 100 and a medial moldable bridge 120, so as to preventthem from any longitudinal or rotational movement with respect to thebone 150 and with respect to each other. The moldable bridges 100 and120 may be made of fast hardening acrylic materials, such as acrylicdental paste, and together with the pins they create a frame having aweight of no more than 1 gr.

FIGS. 18 to 21 schematically illustrate a process of a creation of theCSD in a femur of a mouse 27.

FIGS. 18A and 18B demonstrate the first stage of the process. This stageis temporal positioning and fixation of the limb 21 (comprising of thefemur 20 and the tibia 22) of the mouse with a semi-rigid silicon mold23. The mold 23 is soft and hardens slowly, thereby maintaining the limb21 in a desired position.

FIG. 19 demonstrates the second stage of the process. This stage ispercutaneous insertion of pins 31 (e.g. commercial needles 25G) from thelateral side 30 to the medial side 32 of the femur 20 through holes (notshown that were manually drilled by a drill of corresponding diameter(0.3 mm) from each side of the femur 20. After the insertion of the pins31, they are first fixated with the lateral moldable bridge 100 at thelateral side 30 of the femur 20. Then, the silicon mold 23 is removedand the pins are fixated with the medial moldable bridge 120 at themedial side 32 of the femur 20.

FIG. 20 demonstrates the third stage of the process wherein the pins 31are already fixated with both moldable bridges 100 and 120 and thesilicon mold 23 is removed.

FIG. 21 demonstrates the last stage of the process is inducing the CSD.When the pins 31 are already fixed over the femur 20, the CSD 33 ofabout 2 mm is induced between the pins 31.

FIGS. 19 and 20 illustrate other embodiments of the external fixationsystem 110 for creating a CSD 60 in a long bone 62. The system 110comprises a first couple of pins 61 a and 61 b fixated with firstmoldable bridges 63, and a second couple of pins 65 a and 65 b (shown inFIG. 19B) fixated with second moldable bridges 67. The pins 61 a and 65a are positioned at the right side of the CSD 60, and the pins 61 b and65 b are positioned at the left side of the CSD 60. As shown in FIG.19A, the first couple of pins 61 a and 61 b is parallel to the z axis,so as the bridges 63 are parallel to the y axis (which is the long axisof the bone 62). The second couple of pins 65 a and 65 b is parallel tothe x axis, so as the bridges 67 are parallel to the axis y of the bone.The couples of pins are not necessarily have to be parallel to one ofthe main axes x, y or z, as shown in FIG. 19B, in which the secondcouple of pins 65 a and 65 b is positioned in a certain angle withrespect to the x-y plane.

After the fixation process is completed, the CSD of about 3.5 mm isinduced between the pins using a drill motor.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the present invention.

To the extent that section headings are used, they should not beconstrued as necessarily limiting.

References

Aggarwal S, Pittenger M R 2005 Human mesenchymal stem cells modulateallogeneic immune cell responses. Blood. 105:1815-22.

Bielby R C, Boccaccini A R, Polak J M, Buttery L D. 2004 In vitrodifferentiation and in vivo mineralization of osteogenic cells derivedfrom human embryonic stem cells. Tissue Eng. 10:1518-25.

Bruder S P, Kraus K H, Goldberg V M, Kadiyala S. 1998a The effect ofimplants loaded with autologous mesenchymal stem cells on the healing ofcanine segmental bone defects. J Bone Joint Surg Am. 80:985-96.

Bruder S P, Kurth A A, Shea M, Hayes W C, Jaiswal N, Kadiyala S. 1998bBone regeneration by implantation of purified, culture-expanded humanmesenchymal stem cells. J Orthop Res. 16:155-62.

Buttery L D, Bourne S, Xynos J D, Wood H, Hughes F J, Hughes S P,Episkopou V, Polak J M. 2001 Differentiation of osteoblasts and in vitrobone formation from murine embryonic stem cells. Tissue Eng. 7:89-99.

Chakrabarti S, Mautner V, Osman H, Collingham K E, Fegan C D, Klapper PE, Moss P A, Milligan D W. 2002 Adenovirus infections followingallogeneic stem cell transplantation: incidence and outcome in relationto graft manipulation, immunosuppression, and immune recovery. Blood.100:1619-27.

Cinotti G, Patti A M, Vulcano A, Della Rocca C, Polveroni G, GiannicolaG, Postacchini F. 2004 Experimental posterolateral spinal fusion withporous ceramics and mesenchymal stem cells. J Bone Joint Surg Br.86:135-42.

Choi K, Kennedy M, Kazarov A, Papadimitriou J C, Keller G. 1998 A commonprecursor for hematopoietic and endothelial cells. Development.125:725-32.

Cohen Y, Nagler A. 2004 Umbilical cord blood transplantation--how, whenand for whom? Blood Rev. 18:167-79.

Gang E J, Hong S H, Jeong J A, Hwang S H, Kim S W, Yang I H, Ahn C, HanH, Kim H. 2004 In vitro mesengenic potential of human umbilical cordblood-derived mesenchymal stem cells. Biochem Biophys Res Commun.321:102-8.

Gerasimov IuV, Fridenshtein AIa, Chailakhian R K, Shishkova V V. 1986Differentiation potentials of clonal strains of bone marrow fibroblasts.Biull Eksp Biol Med. 101:717-9.

Gotoh Y, Hiraiwa K, Nagayama M. 1990 In vitro mineralization ofosteoblastic cells derived from human bone. Bone Miner. 8:239-50.

Hamaguchi I, Huang XL, Takakura N, Tada J, Yamaguchi Y, Kodama H, SudaT. 1999 In vitro hematopoietic and endothelial cell development fromcells expressing TEK receptor in murine aorta-gonad-mesonephros region.Blood. 93:1549-56.

He Z, Huang S, Li Y, Zhang Q. 2002 Human embryonic stem cell linespreliminarily established in China. Zhonghua Yi Xue Za Mi. 82:1314-8.

Hofmann G O, Kirschner M H, Gonschorek O, Buhren V. 1998 Bridging longbone and joint defects with allogeneic vascularized transplants.Langenbecks Arch Chir Suppl Kongressbd. 115:1285-7.

Horwitz E M, Prockop D J, Fitzpatrick L A, Koo W W, Gordon P L, Neel M,Sussman M, Orchard P, Marx J C, Pyeritz R E, Brenner M K. 1999Transplantability and therapeutic effects of bone marrow-derivedmesenchymal cells in children with osteogenesis imperfecta. Nat Med.5:309-13.

Horwitz E M, Prockop D J, Gordon P L, Koo W W, Fitzpatrick L A, Neel MD, McCarville M E, Orchard P J, Pyeritz R E, Brenner M K. 2001 Clinicalresponses to bone marrow transplantation in children with severeosteogenesis imperfecta. Blood. 97:1227-31.

Horwitz E M, Gordon P L, Koo W K, Marx J C, Neel M D, McNall R Y, MuulL, Hofmann T. 2002 Isolated allogeneic bone marrow-derived mesenchymalcells engraft and stimulate growth in children with osteogenesisimperfecta: Implications for cell therapy of bone. Proc Natl Acad SciUSA. 99:8932-7.

Hovatta O, Mikkola M, Gertow K, Stromberg A M, Inzunza J, Hreinsson J,Rozell B, Blennow E, Andang M, Ahrlund-Richter L. 2003 A culture systemusing human foreskin fibroblasts as feeder cells allows production ofhuman embryonic stem cells. Hum Reprod. 18:1404-9.

Jaiswal N, Haynesworth S E, Caplan A I, Bruder S R 1997 Osteogenicdifferentiation of purified, culture-expanded human mesenchymal stemcells in vitro. J Cell Biochem. 64:295-312.

Johnson D R. 2000 Differential expression of human majorhistocompatibility class I loci: HLA-A, -B, and -C. Hum Immunol.61:389-96.

Kadiyala S, Young R G, Thiede M A, Bruder S R 1997 Culture expandedcanine mesenchymal stem cells possess osteochondrogenic potential invivo and in vitro. Cell Transplant. 6:125-34.

Kawaguchi H, Hirachi A, Hasegawa N, Iwata T, Hamaguchi H, Shiba H,Takata T, Kato Y, Kurihara H. 2004 Enhancement of periodontal tissueregeneration by transplantation of bone marrow mesenchymal stem cells. JPeriodontol. 75:1281-7.

Koc O N, Peters C, Aubourg P, Raghavan S, Dyhouse S, DeGasperi R,Kolodny E H, Yoseph Y B, Gerson S L, Lazarus H M, Caplan A I, Watkins PA, Krivit W. 1999 Bone marrow-derived mesenchymal stem cells remainhost-derived despite successful hematopoietic engraftment afterallogeneic transplantation in patients with lysosomal and peroxisomalstorage diseases. Exp Hematol. 27:1675-81.

Krampera M, Glennie S, Dyson J, Scott D, Laylor R, Simpson E, Dazzi F.2003 Bone marrow mesenchymal stem cells inhibit the response of naiveand memory antigen-specific T cells to their cognate peptide. Blood.101:3722-9.

Lane J M, Yasko A W, Tomin E, Cole B J, Waller S, Browne M, Turek T,Gross J. 1999 Bone marrow and recombinant human bone morphogeneticprotein-2 in osseous repair. Clin Orthop. 361:216-27.

Lee O K, Kuo T K, Chen W M, Lee K D, Hsieh S L, Chen T H. 2004 Isolationof multipotent mesenchymal stem cells from umbilical cord blood. Blood.103:1669-75.

Lee W Y, Cho S W, Oh E S, Oh K W, Lee J M, Yoon K H, Kang M I, Cha B Y,Lee K W, Son H Y, Kang S K, Kim C C. 2002 The effect of bone marrowtransplantation on the osteoblastic differentiation of human bone marrowstromal cells. J Clin Endocrinol Metab. 87:329-35.

Lewandrowski K U, Gresser J D, Wise D L, Trantol D J. 2000 Bioresorbablebone graft substitutes of different osteoconductivities: a histologicevaluation of osteointegration of poly(propylene glycol-co-fumaricacid)-based cement implants in rats. Biomaterials. 21:757-64.

Maitra B, Szekely E, Gjini K, Laughlin M J, Dennis J, Haynesworth S E,Koc ON. 2004 Human mesenchymal stem cells support unrelated donorhematopoietic stem cells and suppress T-cell activation. Bone MarrowTransplant. 33:597-604.

Mitchell K E, Weiss M L, Mitchell B M, Martin P, Davis D, Morales L,Helwig B, Beerenstrauch M, Abou-Easa K, Hildreth T, Troyer D, MedicettyS. 2003 Matrix cells from Wharton's jelly form neurons and glia. StemCells. 21:50-60.

Mizuno H, Hyakusoku H. 2003 Mesengenic potential and future clinicalperspective of human processed lipoaspirate cells. J Nippon Med Sch.70:300-6.

Murohara T, Ikeda H, Duan J, Shintani S, Sasaki K, Eguchi H, Onitsuka I,Matsui K, Imaizumi T. 2000 Transplanted cord blood-derived endothelialprecursor cells augment postnatal neovascularization. J Clin Invest.105:1527-36.

Nade S, Armstrong L, McCartney E, Baggaley B. 1983 Osteogenesis afterbone and bone marrow transplantation. The ability of ceramic materialsto sustain osteogenesis from transplanted bone marrow cells: preliminarystudies. Clin Orthop Relat Res. 181:255-63.

Neppert J, Nunez G, Stastny P. 1984 HLA-A, B, C; -DR; -MT, -MB, and SBantigens on unstimulated human endothelial cells. Tissue Antigens.24:40-7.

Nilsson S K, Dooner M S, Weier H U, Frenkel B, Lian J B, Stein G S,Quesenberry P J. 1999 Cells capable of bone production engraft fromwhole bone marrow transplants in nonablated mice. J Exp Med. 189:729-34.

Niemeyer P, Seckinger A, Simank H G, Kasten P, Sudkamp N, Krause U. 2004Allogenic transplantation of human mesenchymal stem cells for tissueengineering purposes: an in vitro study. Orthopade. 33:1346-53.

Ohgushi H, Okumura M, Tamai S, Shors E C, Caplan A L 1990 Marrow cellinduced osteogenesis in porous hydroxyapatite and tricalcium phosphate:a comparative histomorphometric study of ectopic bone formation. JBiomed Mater Res. 24:1563-70.

Pereira R F, Halford K W, O'Hara M D, Leeper D B, Sokolov B P, Pollard MD, Bagasra O, Prockop D J. 1995 Cultured adherent cells from marrow canserve as long-lasting precursor cells for bone, cartilage, and lung inirradiated mice. Proc Natl Acad Sci USA. 92:4857-61.

Petersen B E, Bowen W C, Patrene K D, Mars W M, Sullivan A K, Murase N,Boggs S S, Greenberger J S, Goff J R 1999 Bone marrow as a potentialsource of hepatic oval cells. Science. 284:1168-70.

Ringe J, Kaps C, Schmitt B, Buscher K, Bartel J, Smolian H, Schultz O,Burmester G R, Haupl T, Sittinger M. 2002 Porcine mesenchymal stemcells. Induction of distinct mesenchymal cell lineages. Cell Tissue Res.307:321-7.

Schmidt G M, Horak D A, Niland J C, Duncan S R, Forman S J, Zaia J A.1991 A randomized, controlled trial of prophylactic ganciclovir forcytomegalovirus pulmonary infection in recipients of allogeneic bonemarrow transplants; The City of Hope-Stanford-Syntex CMV Study Group. NEngl J Med. 1991 324:1005-11.

Shahgasempour S, Woodroffe S B, Garnett H M. 1998 Modulation of HLAclass I antigen and ICAM-2 on endothelial cells after in vitro infectionwith human cytomegalovirus. Immunol Cell Biol. 76:217-21

Shang Q, Wang Z, Liu W, Shi Y, Cui L, Cao Y. 2001 Tissue-engineered bonerepair of sheep cranial defects with autologous bone marrow stromalcells. J Craniofac Surg. 12:586-93;

Sottile V, Thomson A, McWhir J. 2003 In vitro osteogenic differentiationof human ES cells. Cloning Stem Cells. 5:149-55.

Sun S, Guo Z, Xiao X, Liu B, Liu X, Tang P H, Mao N. 2003 Isolation ofmouse marrow mesenchymal progenitors by a novel and reliable method.Stem Cells. 21:527-35.

Suparno C, Milligan D W, Moss P A, Mautner V. 2004 Adenovirus infectionsin stem cell transplant recipients: recent developments in understandingof pathogenesis, diagnosis and management. Leuk Lymphoma. 45:873-85.

Theunissen K, Verfaillie C M. 2005 A multifactorial analysis ofumbilical cord blood, adult bone marrow and mobilized peripheral bloodprogenitors using the improved ML-IC assay. Exp Hematol. 33:165-72.

Valimaki M J, Kinnunen K, Volin L, Tahtela R, Loyttyniemi E, Laitinen K,Makela P, Keto P, Ruutu T. 1999 A prospective study of bone loss andturnover after allogeneic bone marrow transplantation: effect of calciumsupplementation with or without calcitonin. Bone Marrow Transplant.23:355-61.

Wakitani S, Takaoka K, Hattori T, Miyazawa N, Iwanaga T, Takeda S,Watanabe T K, Tanigami A. 2003 Embryonic stem cells injected into themouse knee joint form teratomas and subsequently destroy the joint.Rheumatology (Oxford). 42:162-5.

Wang H S, Hung S C, Peng S T, Huang C C, Wei H M, Guo Y J, Fu Y S, Lai MC, Chen C C. 2004 Mesenchymal stem cells in the Wharton's jelly of thehuman umbilical cord. Stem Cells. 22:1330-7.

Werntz J R, Lane J M, Burstein A H, Justin R, Klein R, Tomin E. 1996Qualitative and quantitative analysis of orthotopic bone regeneration bymarrow. J Orthop Res. 14:85-93.

Winston D J, Ho W G, Champlin R E. 1990 Cytomegalovirus infections afterallogeneic bone marrow transplantation. Rev Infect Dis. 12 Suppl7:S776-92.

Yoshikawa T, Ohgushi H. 1999 Autogenous cultured bone graft-bonereconstruction using tissue engineering approach. Ann Chir Gynaecol.88:186-92.

Zuk P A, Zhu M, Mizuno H, Huang J, Futrell J W, Katz A J, Benhaim P,Lorenz H P, Hedrick M H. 2001 Multilineage cells from human adiposetissue: implications for cell-based therapies. Tissue Eng. 7:211-28.

Zuk P A, Zhu M, Ashjian P, De Ugarte D A, Huang J I, Mizuno H, Alfonso ZC, Fraser J K, Benhaim P, Hedrick M H. 2002 Human adipose tissue is asource of multipotent stem cells. Mol Biol Cell. 13:4279-95.

zur Nieden N I, Kempka G, Ahr H J. 2003 In vitro differentiation ofembryonic stem cells into mineralized osteoblasts. Differentiation.71:18-27.

1. A prosthetic implant comprising a biocompatible three-dimensionalscaffold and at least two cell types selected from the group consistingof osteoblasts, osteoclasts, chondrocytes, and endothelial cells orprogenitors thereof.
 2. A method of producing a prosthetic implant,comprising isolating, expanding and co-cultivating at least two celltypes selected from the group consisting of osteoblasts, osteoclasts,chondrocytes, and endothelial cells or progenitors thereof on a3-dimensional scaffold.
 3. The method of claim 2, wherein said method isperformed in a flow system.
 4. A method of producing a prostheticimplant, comprising isolating, expanding and co-cultivating at least twocell types selected from the group consisting of osteoblasts,osteoclasts, chondrocytes, and endothelial cells or progenitors thereofon a 3-dimensional scaffold in a flow system.
 5. The method of any ofclaims 1-4, wherein said at least two cell types comprises three celltypes.
 6. The method of any of claims 1 to 5, wherein said at least twocell types are selected from the group consisting of osteoblasts,osteoclasts and endothelial cells.
 7. A prosthetic implant comprising abiocompatible three-dimensional scaffold and at least three cell types,including at least one of osteoblasts or osteoclasts, or progenitorsthereof.
 8. The implant or method of any of the preceding claims,wherein said cells are grown to a density of at least 10⁶ cells/ml. 9.The implant or method of any of the preceding claims, wherein said cellsare grown to a density of at least 5×10⁶ cells/ml.
 10. The implant ormethod of any of the preceding claims, wherein said cells are grown to adensity of at least 10⁷ cells/ml.
 11. The implant or method of any ofclaims 1 to 10, wherein said progenitors are selected from the groupconsisting of mesenchymal stem cells, hematopoietic stem cells, andangioblasts.
 12. The implant or method of any of claims 1-11, whereinsaid at least two cell types are obtained from a source selected fromthe group consisting of an autologous source, a syngeneic source, and anallogeneic source, or a combination thereof.
 13. The implant or methodof claim 12, wherein said source is selected from the group consistingof bone marrow, placenta, adipose tissue, cord blood, cord vein,peripheral blood, mobilized peripheral blood, and embryonic stem cells.14. The implant or method of any of claims 1 to 13, wherein saidthree-dimensional scaffold comprises a material selected from the groupconsisting of an organic material, a polymer, a ceramic material and ametallic material.
 15. The implant or method of claim 14, wherein saidorganic material comprises a bone particle selected from the groupconsisting of a dry bone particle, a frozen bone particle and adematerialized bone particle.
 16. The implant or method of claim 14,wherein said polymer or ceramic material is selected from the groupconsisting of aliphatic polyesters, poly (amino acids), co-poly(ether-esters), polyalkylenes oxalates, polyamides, tyrosine derivedpolycarbonates, poly (iminocarbonates), polyorthoesters, polyoxaesters,polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, bioactive glass, calcium phosphatederivatives, calcium sulfate derivatives, calcium hydroxyapeptite,silicate matrices, hydroxyapeptite, hyalauronic acid, beta-3 calciumphosphate, cross-linked collage fibers, resin based materials, fibrin,fibronectin, collagen, ECM and combinations thereof.
 17. The implant ormethod of claim 14, wherein said polymer comprises a biodegradablepolymer selected from the group consisting of a solid polymer, semisolidpolymers and a gel like polymer.
 18. The implant or method of claim 17,wherein said biodegradable polymer is selected from the group consistingof poly caprolactone, poly-glycolic acid, poly-lactic acid, poly lacticco-glycolic, poly tartonic acid, fibrin, fibronectin, collagen, ECM orcopolymers thereof, and combinations thereof.
 19. The implant or methodof any of the preceding claims, wherein said three-dimensional scaffoldhas a pore size in the range of from about 50 microns to about 2000microns.
 20. The implant or method of any of the preceding claims,wherein said three-dimensional scaffold comprises a coating layer. 21.The implant or method of claim 20, wherein said coating layer comprisesa material selected from the group consisting of poly-D-lysine,collagen, fibronectin ECM and hydrogel, or a combination thereof. 22.Use of the implant or method of any of the preceding claims for supportof high density cell growth.
 23. The use of claim 22, wherein said cellgrowth is performed in a flow system.
 24. The use of claim 23, whereinsaid flow system comprises a bioreactor.
 25. The use of claim 24,wherein said bioreactor comprises a growth matrix.
 26. The use of claim25, wherein said growth matrix is in sheet form.
 27. The use of claim26, wherein said sheet comprises a non-woven fiber.
 28. The use of anyof claim 26 or 27, wherein said sheet comprises an open-pored foampolymer.
 29. The use of any of claims 26 to 28, wherein a thickness ofsaid sheet is in the range of from about 50 to about 2000 microns. 30.The use of any of claims 26 to 29, wherein said sheet comprises poreshaving a diameter in the range of from about 10 microns to about 500microns.
 31. The use of claim 30, wherein said diameter is in the rangeof from about 0.5 microns to about 20 microns.
 32. The use of claim 31,wherein said diameter is in the range of from about 10 microns to about15 microns.
 33. The implant, use or method of any of the precedingclaims, wherein said three-dimensional scaffold is pre-shaped forimplant into a bone lesion.
 34. The implant, use or method of any of thepreceding claims, wherein said isolating comprises preparing samples ofbone marrow, fat tissue, placenta or cord vein; obtaining a suspension;centrifuging said suspension; and collecting precipitated cells.
 35. Themethod of any of claims 4-6, wherein said co-cultivation comprisessubjecting said cells to an osteogenic stimulus.
 36. The method of claim35, wherein said osteogenic stimulus comprises contacting the cells witha molecule selected from the group consisting of dexamethasone, sodiumβ-glycerophosphate, 1,25 dihydroxycholecalciferol calcitriol, andL-ascorbic acid-2-phosphate (10-500 nM).
 37. The method of claim 36,wherein a concentration of said dexamethasone is in the range of fromabout 10 to about 200 nM.
 38. The method of claim 36, wherein aconcentration of said sodium β-glycerophosphate is in the range of fromabout 5 to about 25 mM.
 39. The method of claim 36, wherein aconcentration of said 1,25 dihydroxycholecalciferol calcitriol is in therange of from about 5 to about 50 nM.
 40. The method of any of claim 4-6or 35-39, wherein said co-cultivation is carried out in a mediumcomprising at least one of a growth factor and a cytokine.
 41. Themethod of claim 40, wherein said growth factor or cytokine is selectedfrom the group consisting of transforming growth factor beta (TGF beta),insulin-like growth factor-1 and 2 (IGF-1 and 2), PDGF (platelet derivedgrowth factor), osteogenic protein-1 (OP-1), fibroblast growth factor(FGF), FGF-2, FGF-9, FGF-10, PTH (parathyroid hormone), PRP (plateletrich plasma), EGF (epidermal growth factor), VEGF (vascular endothelialgrowth factor) and a Bone Morphogenic Protein (BMP) family member, or acombination thereof.
 42. The method of claim 41, wherein said bonemorphogenic protein is selected from the group consisting of BMP-2,BMP-4 and BMP-7.
 43. The method of claim 36, wherein said osteogenicstimulus comprises exposure to shear forces.
 44. The method of any ofclaims 34-43, wherein said cells in said suspension are contacted with aplurality of enzymes, including at least collagenase.
 45. The implant,method or use of any of the preceding claims for use in an applicationselected from the group consisting of bone replacement, boneaugmentation, dental application, combined bone/cartilage application,plastic surgery applications, bone or cartilage repair, cartilagereplacement, orthopedic applications and other hard tissue applications.46. The implant, method or use of any of the preceding claims, whereinsaid implant is cryopreserved and thawed prior to use.
 47. An allograftor autograft plug comprising a biocompatible scaffold, wherein a firstside of said scaffold comprises a coating of cartilage tissue, andwherein a second side of said scaffold comprises a layer comprisingosteoblasts, and chondrocytes or progenitors thereof, prepared accordingto any of the above claims.
 48. A method of producing an allograft orautograft plug, comprising: Coating a first side of a biocompatiblescaffold with a layer of cartilage tissue; Isolating, expanding andco-cultivating osteoblasts and chondrocytes, or progenitors thereof, ona second side of said biocompatible scaffold, according to any of theabove claims.
 49. The method of claim 48, wherein said allograft plug isfurther cryopreserved.
 50. Use of the allograft plug of claim 49 fortransplantation into a patient in need thereof, wherein said allograftis thawed prior to transplantation.
 51. The use of claim 50, whereinsaid transplantation comprises a press fit technique.
 52. An externalfixation system for creating a defect in a long bone of an animal, thesystem comprising a frame composed of: at least two pins forpercutaneous insertion from the lateral to the medial side of said bonespaced apart in a distance greater than the length of the defect; atleast one moldable bridge for fixation of said pins with respect to eachother and over said bone in a manner to prevent the pins from anylongitudinal or rotational displacement with respect to said bone andwith respect to each other.
 53. A method for creating a defect in a longbone of an animal, the method comprising: providing an external fixationsystem comprising a frame composed of at least two pins and at least onemoldable bridge; temporarily positioning and fixation of a limb of theanimal to allow a desired position thereof; percutaneous insertion ofsaid pins from the lateral to the medial side of said bone in a distancegreater than the length of the defect; fixation of said pins with saidmoldable bridge with respect to each other and over said bone in amanner to prevent the pins from any longitudinal or rotationaldisplacement with respect to said bone and with respect to each other;induction of the defect.
 54. A kit for creating a defect in a long boneof an animal, the kit comprising: a frame composed of at least two pinsfor percutaneous insertion from the lateral to the medial side of saidbone spaced apart in a distance greater than the length of the defect;and at least one moldable bridge for fixation of said pins with respectto each other and over said bone in a manner to prevent the pins fromany longitudinal or rotational displacement with respect to said boneand with respect to each other; a mold made of flexible material fortemporarily positioning and fixation of a limb of the animal to allow adesired position thereof.